Skin epidermis is derived from the pluripotent ectoderm. In an ectodermal cell's path to become interfollicular epidermis, it faces at least three critical decisions: 1) not to become a neural cell, 2) not to become an appendage cell, 3) to self-renew (as a stem or progenitor cell normally does), or to cease proliferation and terminally differentiate into a highly specialized cell that carries an essential protective function for the organism's survival. How are these choices made? What genes or genetic pathways govern or modulate these choices? What happens if these genes/pathways go awry? The long-term goal of my research is to use a multidisciplinary approach to address these important questions that will impact the understanding of not only stem cell proliferation and differentiation in skin, but also the mechanisms of epithelial development and differentiation in general. This proposal focuses on addressing the role of two mouse Ovo genes, Ovol1 and Ovol2, encoding zinc finger transcription factors, in epidermal specification, proliferation, and differentiation. There are three specific aims: 1) Characterize the role of Ovol2 in embryonic stem (ES) cell proliferation, ectodermal differentiation, and epidermal specification. We will generate ES cells deficient in Ovol2, and analyze their proliferation and survival in vitro. We will adapt an in vitro culture system that allows the differentiation of ES cells into epidermal cells, and monitor epidermal differentiation of wild-type and Ovol2-deficient ES cells. We will perform chimeric mouse analysis to examine the intrinsic ability of Ovol2-deficient ES cells in contributing to the skin epidermis. 2) Characterize the role of Ovol2 in epidermal proliferation and differentiation. We will examine the function of Ovol2 in epidermis through the generation and analysis of epidermis-specific Ovol2 knockout mice, particularly testing the hypothesis that Ovol2 is required for self- renewal and/or proliferation of epidermal stem/progenitor cells. 3) Investigate the mechanism of Ovol1 function in gene expression and growth arrest. We will perform biochemical studies to test the hypothesis that Ovol1 protein directly represses c-Myc and Id2 expression by competing with c-Myb for binding and by recruiting HDACs to their promoters, thereby facilitating growth arrest. Furthermore, we will employ a transgenic "dominant positive" approach to further address the in vivo role of Ovoll in the regulation of proliferation/differentiation in developing epidermis.